The present invention relates to the cloning and expression of a human gene encoding a protein having deoxycytidyl (dCMP) transferase activity. The present invention also provides methods for expressing the gene, and cancer treating methods using inhibitors to the gene.
DNA damage can lead to mutations during replication. In the yeast S. cerevisiae, it appears that the majority of induced mutations are generated through the damage-induced mutagenesis pathway (1,2). The required yeast genes in this pathway include: RAD6, RAD18, REV1, REV3, REV6, REV7, and NGM2 (1-7), most of which have been isolated by gene cloning. As expected, inactivating these mutagenesis genes dramatically decreases the mutation frequency following DNA damage (3,8).
Rad6 is a ubiquitin-conjugating enzyme (9) and forms a complex with Rad18 (10-12). It has been proposed that this complex may play an important role in the initial steps of the damage-induced mutagenesis pathway (10). Rev3 protein is a DNA polymerase (DNA polymerase xcex6) capable of translesion DNA synthesis (13). In contrast to the replicative DNA polymerases, deletion of the yeast REV3 gene does not lead to lethality (1). Hence, this polymerase is specifically required for damage-induced mutagenesis in yeast. Rev1 belongs to the UmuC family of proteins (14). It possesses a deoxycytidyl (dCMP) transferase activity in a template-dependent reaction, which can efficiently insert a dCMP opposite a template AP (apurinic/apyrimidinic) site (15). Yeast Rad30, an E. coli DinB homologue, is another member of the UmuC family (14,16,17). However, unlike Rev1, Rad30 is not a component of the damage-induced mutagenesis pathway, but appears to be involved in a novel error-free lesion bypass mechanism (16,17). Most recently, Rad30 was shown to be a nonessential DNA polymerase (pol xcex7) capable of error-free translesion DNA synthesis opposite a TT dimer in vitro (18). Apparently, the UmuC family of proteins are involved in different mechanisms in the damage tolerance response to unrepaired DNA lesions during replication.
It is only very recently that the damage-induced mutagenesis pathway in humans has been investigated. Two human homologues of the yeast RAD6 gene have been identified: HHR6A and HHR6B (19,20). Additionally, hREV3 has been isolated as the human homologue of the yeast mutagenic DNA polymerase xcex6 (21,22). Thus, it is most likely that a damage-induced mutagenesis pathway similar to that in yeast is operational in humans. Given the genetic complexity of the yeast mutagenesis pathway, it is certain that more human mutagenesis genes remain to be identified. Since mutations are the building blocks of human cancers, understanding the damage-induced mutagenesis pathway in humans is a key to the understanding of carcinogenesis. Isolating the human mutagenesis genes and elucidating the activities of these gene products are essential steps in these studies.
There is always a need for more effectively diagnosing, preventing and treating cancer. This applies to the determination of polypeptides that are involved in causing mutations that lead to the formation of tumors and further mutations that cause metastasis to occur.
Applicants have isolated a full-length cDNA representing the homologue of the yeast Rev1 mutagenesis protein. Applicants also determined the chromosomal location of the human REV1 gene and demonstrated its ubiquitous expression in various human tissues. Furthermore, Applicants have demonstrated that the human REV1 protein is a dCMP transferase capable of inserting a dCMP opposite a template AP site.
The present invention also relates to antibodies, including monoclonal or polyclonal antibodies, and antibody fragments that have specific interaction with epitopes present on hREV1. The present invention is also directed to methods of preventing, treating, or ameliorating a disease condition or disorder in an individual comprising the step of administering a therapeutically effective amount of hREV1 protein or its inhibitor or activator to the individual. The present invention is also directed to methods or protocols in treatment or prevention of a disease or disorder based on the gene and gene product described in the present application.
The present invention is related to an isolated nucleic acid molecule that encodes:
a polynucleotide which encodes the polypeptide set forth in Table 1;
a polynucleotide which encodes a variant of the polypeptide set forth in Table 1 wherein said variant has a deoxycytidyl transferase activity;
a polynucleotide which encodes a homologous variant of said polypeptide set forth in Table 1 having less than about 750 amino acid changes; or
a polynucleotide sequence which hybridizes to the polynucleotide of Table 1 under the following conditions:
prehybridize the membrane in solution of 0.25M sodium phosphate, 0.25 M NaCl, 1 mM EDTA, 5%SDS and 50% formamide for 1 to 4 hours at 42xc2x0 C. and then hybridize in the same solution with denatured labeled DNA probe and at 42xc2x0 C. for overnight. After hybridization, wash the membrane with 0.2xc3x97SSPE, 0.1% SDS at 42xc2x0 C. for 30 minutes and then wash in more stringent condition with 0.1xc3x97SSPE, 0.05%SDS at higher temperature, for example, at 55xc2x0 C. for 30 minutes. Preferably, the polynucleotide is a DNA. More preferably, the DNA comprises a DNA encoding the polypeptide sequence of Table 1.
The present invention is further directed to a vector, comprising:
a replicable vector; and
the above-describe polynucleotide inserted into said vector.
The present invention is also directed to polypeptides encoded by the nucleic acid described above.
The present invention is related to a pharmaceutical composition comprising:
a therapeutically effective amount of an inhibitor to the above described polypeptide; and
a pharmaceutically acceptable carrier or diluent.
It is an object of the invention to provide a method of preventing tumor formation, comprising administering to a person in need thereof, a prophylactic amount of an inhibitor to human deoxycytidyl transferase. Preferably, the inhibitor blocks transcription or translation of the deoxycytidyl transferase gene. Also preferred is that the inhibitor blocks activity of the deoxycytidyl transferase protein by binding to the protein, in which case the preferred inhibitor is an antibody, more preferably, a monoclonal antibody.
Another object of the invention is to provide a method for treating or slowing metastasis of a tumor, comprising administering to a person in need thereof, a therapeutically effective amount of an inhibitor to human deoxycytidyl transferase.
Yet another object of the present invention is to provide a method for preventing mutations in a person, comprising administering to a person in need thereof, a therapeutically effective amount of an inhibitor to human deoxycytidyl transferase.
Table 1 describes the nucleotide and the deduced amino acid sequences of the human REV1 (hREV1) gene. Nucleotide sequence of the full-length hREV1 is shown in upper case letters while the 5xe2x80x2 and the 3xe2x80x2 nontranslated regions are shown in lower case letters. The deduced amino acid sequence is shown by the single letter symbols of amino acids. A mini open reading frame upstream of the hREV1 open reading frame is shown by the underline. The bold-type indicates the putative polyadenylation signal.